An Overview of the Management of Mansonellosis

Mansonellosis is caused by three filarial parasite species from the genus Mansonella that commonly produce chronic human microfilaraemias: M. ozzardi, M. perstans and M. streptocerca. The disease is widespread in Africa, the Caribbean and South and Central America, and although it is typically asymptomatic it has been associated with mild pathologies including leg-chills, joint-pains, headaches, fevers, and corneal lesions. No robust mansonellosis disease burden estimates have yet been made and the impact the disease has on blood bank stocks and the monitoring of other filarial diseases is not thought to be of sufficient public health importance to justify dedicated disease management interventions. Mansonellosis´s Ceratopogonidae and Simuliidae vectors are not targeted by other control programmes and because of their small size and out-door biting habits are unlikely to be affected by interventions targeting other disease vectors like mosquitoes. The ivermectin and mebendazole-based mass drug administration (iMDA and mMDA) treatment regimens deployed by the WHO´s Elimination of Neglected Tropical Diseases (ESPEN) programme and its forerunners have, however, likely impacted significantly on the mansonellosis disease burden, principally by reducing the transmission of M. streptocerca in Africa. The increasingly popular plan of using iMDA to control malaria could also affect M. ozzardi parasite prevalence and transmission in Latin America in the future. However, a potentially far greater mansonellosis disease burden impact is likely to come from shortcourse curative anti-Wolbachia therapeutics, which are presently being developed for onchocerciasis and lymphatic filariasis treatment. Even if the WHO´s ESPEN programme does not choose to deploy these drugs in MDA interventions, they have the potential to dramatically increase the financial and logistical feasibility of effective mansonellosis management.There is, thus, now a fresh and urgent need to better characterise the disease burden and ecoepidemiology of mansonellosis so that effective management programmes can be designed, advocated for and implemented.We would like to express our special thanks to María Belén García Fernández for helping us to draw the life-cycle of Mansonella species. JLC and SLBL also gratefully acknowledge support from the Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM; 062.01282/2018 and 002.00200/2019). And JLC would like to acknowledge support he receives from a Conselho Nacional de Desenvolvimento Científica e Tecnológico (CNPq) productivity grant. THTT is funded by a Sara Borrell contract from the Instituto de Salud Carlos III.S

Nested multiplex PCR for identification and detection of human Plasmodium species including Plasmodium knowlesi

Objective: To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria. Methods: In this study the nested multiplex malaria PCR was redesigned, targeting the 18S rRNA gene, to identify the fifth human Plasmodium species, Plasmodium knowlesi, together with the other human Plasmodium (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives. The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms. The technique showed high sensitivity (100%) and specificity (96%) when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos Ⅲ and a published real-time PCR malaria assay. Conclusions: The technique designed is an economical, sensitive and specific alternative to current diagnosis methods. Furthermore, the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.This work was supported by AESI-ISCⅢ grant number PI14CⅢ/00014.S

Usefulness of a commercial LAMP assay for detection of malaria infection, including Plasmodium knowlesi cases, in returning travelers in Spain

Objetive: Main malaria diagnosis is based on microscopic examination combined with rapid diagnostic tests. Both methods have low sensitivity and specificity. Loop-mediated isothermal amplification techniques have shown a sensitivity similar to PCR but with lower times of performance. This study aimed to assess a commercial LAMP for the diagnosis of malaria (Alethia® Malaria) against the Nested-Multiplex-Malaria PCR, including the analytical sensitivity and the operational characteristics. Results: One hundred five samples out of 114 rendered valid results, obtaining 85 positive samples and 18 negative samples with an agreement of 98% compared to the reference method with a sensitivity, specificity and kappa coefficient of 98.84%, 94.74% and 0.94 respectively, with only two discrepant samples. The turnaround time was estimated in 1 h and 30 min, with a cost of 32.67€ per determination. The results showed several advantages of the Alethia® Malaria, as it was easy to perform, minimal training requirement and 40 min run. Moreover, it includes an internal control to avoid false negatives. However, it also showed some limitations such as the need for a specific amplification and detection device, the detection of only Plasmodium spp. and a very high price.This work was partially supported by the Spanish Strategic Health Action (AESI-ISCIII) Grant Number PI17CIII/00035. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Alexandra Martín Ramírez is supported by an ISCIII Río Hortega grant.S

Uso de PCR múltiple en el diagnóstico simultáneo de parasitosis

Simposio: Nuevos desarrollos en el diagnóstico de parásitos. XV Congreso Colombiano de Parasitología y Medicina Tropical. 2011N

Mansonellosis: current perspectives

Mansonellosis is a filarial disease caused by three species of filarial (nematode) parasites (Mansonella perstans, Mansonella streptocerca, and Mansonella ozzardi) that use humans as their main definitive hosts. These parasites are transmitted from person to person by bloodsucking females from two families of flies (Diptera). Biting midges (Ceratopogonidae) transmit all three species of Mansonella, but blackflies (Simuliidae) are also known to play a role in the transmission of M. ozzardi in parts of Latin America. M. perstans and M. streptocerca are endemic in western, eastern, and central Africa, and M. perstans is also present in the neotropical region from equatorial Brazil to the Caribbean coast. M. ozzardi has a patchy distribution in Latin America and the Caribbean. Mansonellosis infections are thought to have little pathogenicity and to be almost always asymptomatic, but occasionally causing itching, joint pains, enlarged lymph glands, and vague abdominal symptoms. In Brazil, M. ozzardi infections are also associated with corneal lesions. Diagnosis is usually performed by detecting microfilariae in peripheral blood or skin without any periodicity. There is no standard treatment at present for mansonellosis. The combination therapy of diethylcarbamazine plus mebendazole for M. perstans microfilaremia is presently one of the most widely used, but the use of ivermectin has also been proven to be very effective against microfilariae. Recently, doxycycline has shown excellent efficacy and safety when used as an antimicrobial against endosymbiotic Wolbachia bacteria harbored by some strains of M. perstans and M. ozzardi. Diethylcarbamazine and ivermectin have been used effectively to treat M. streptocerca infection. There are at present no estimates of the disease burden caused by mansonellosis, and thus its importance to many global health professionals and policy makers is presently limited to how it can interfere with diagnostic tools used in modern filarial disease control and elimination programs aimed at other species of filariae.S

Assessment of Commercial Real-Time PCR Assays for Detection of Malaria Infection in a Non-Endemic Setting.

Malaria control and elimination require prompt diagnosis and accurate treatment. Conventional methods such as rapid diagnostic tests (RDTs) and microscopy lack the characteristics to detect low parasitemias, commonly found in asymptomatic parasitemias and/or submicroscopic malaria carriers. On the contrary, molecular methods have higher sensitivity and specificity. This study evaluated the performance of two commercial real-time polymerase chain reaction (PCR) assays, RealStar® Malaria PCR (RealStar-genus) and RealStar Malaria Screen&Type PCR (RealStar-species), compared with the reference Nested Multiplex Malaria PCR, for the detection of the main five Plasmodium species affecting humans. A total of 121 samples were evaluated. Values of sensitivity (98.9% and 97.8%) and specificity (100% and 96.7%) of the RealStar-genus and the RealStar-species assays, respectively, were very good. The limit of detection (LoD) for the RealStar-genus assay showed a mean value of 0.28 parasites/µL with Plasmodium falciparum samples; while, the LoD of the RealStar-species assay ranged from 0.09 parasites/µL for P. vivax to two parasites/µL for P. ovale. The time to complete a diagnosis was established in 4 hours. Our findings showed a very good concordance of both assays compared with the reference method, with a very good analytical sensitivity. RealStar-species assay was able to correctly characterize double and triple infections. Therefore, these RealStar assays have shown to be useful tools in malaria diagnosis in non-endemic countries and even endemic countries, and for malaria control in general, detecting low parasitemias with sensitivity similar to the most sensitive methods as nested PCR, but with lower time to get the results.This work was funded by projects PI14CIII/00014 and PI17CIII/00035 from the Instituto de Salud Carlos III (Ministry of Science and Innovation) and cofounded by the European Regional Development Fund. Altona Diagnostics also partially funded this study by donating the kits and funding the necessary consumables. Alexandra Martin Ramirez is supported by an ISCIII Rio Hortega contract.S

First case of a naturally acquired human infection with Plasmodium cynomolgi

Since 1960, a total of seven species of monkey malaria have been reported as transmissible to man by mosquito bite: Plasmodium cynomolgi, Plasmodium brasilianum, Plasmodium eylesi, Plasmodium knowlesi, Plasmodium inui, Plasmodium schwetzi and Plasmodium simium. With the exception of P. knowlesi, none of the other species has been found to infect humans in nature. In this report, it is described the first known case of a naturally acquired P. cynomolgi malaria in humans.The patient was a 39-year-old woman from a malaria-free area with no previous history of malaria or travel to endemic areas. Initially, malaria was diagnosed and identified as Plasmodium malariae/P. knowlesi by microscopy in the Terengganu State Health Department. Thick and thin blood films stained with 10% Giemsa were performed for microscopy examination. Molecular species identification was performed at the Institute for Medical Research (IMR, Malaysia) and in the Malaria & Emerging Parasitic Diseases Laboratory (MAPELAB, Spain) using different nested PCR methods.Microscopic re-examination in the IMR showed characteristics of Plasmodium vivax and was confirmed by a nested PCR assay developed by Snounou et al. Instead, a different PCR assay plus sequencing performed at the MAPELAB confirmed that the patient was infected with P. cynomolgi and not with P. vivax.This is the first report of human P. cynomolgi infection acquired in a natural way, but there might be more undiagnosed or misdiagnosed cases, since P. cynomolgi is morphologically indistinguishable from P. vivax, and one of the most used PCR methods for malaria infection detection may identify a P. cynomolgi infection as P. vivax.Simian Plasmodium species may routinely infect humans in Southeast Asia. New diagnostic methods are necessary to distinguish between the human and monkey malaria species. Further epidemiological studies, incriminating also the mosquito vector(s), must be performed to know the relevance of cynomolgi malaria and its implication on human public health and in the control of human malaria.The zoonotic malaria cannot be ignored in view of increasing interactions between man and wild animals in the process of urbanization.This work was supported by the Interuniversity Cooperation Programme (AECID grant and A1/035539/11) and IMR/NIH Project NMRR-11-410-9622. THT was financed by a fellowship from the Instituto de Salud Carlos III.S

Plasmodium species differentiation by non-expert on-line volunteers for remote malaria field diagnosis

BACKGROUND: Routine field diagnosis of malaria is a considerable challenge in rural and low resources endemic areas mainly due to lack of personnel, training and sample processing capacity. In addition, differential diagnosis of Plasmodium species has a high level of misdiagnosis. Real time remote microscopical diagnosis through on-line crowdsourcing platforms could be converted into an agile network to support diagnosis-based treatment and malaria control in low resources areas. This study explores whether accurate Plasmodium species identification-a critical step during the diagnosis protocol in order to choose the appropriate medication-is possible through the information provided by non-trained on-line volunteers. METHODS: 88 volunteers have performed a series of questionnaires over 110 images to differentiate species (Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, Plasmodium malariae, Plasmodium knowlesi) and parasite staging from thin blood smear images digitalized with a smartphone camera adapted to the ocular of a conventional light microscope. Visual cues evaluated in the surveys include texture and colour, parasite shape and red blood size. RESULTS: On-line volunteers are able to discriminate Plasmodium species (P. falciparum, P. malariae, P. vivax, P. ovale, P. knowlesi) and stages in thin-blood smears according to visual cues observed on digitalized images of parasitized red blood cells. Friendly textual descriptions of the visual cues and specialized malaria terminology is key for volunteers learning and efficiency. CONCLUSIONS: On-line volunteers with short-training are able to differentiate malaria parasite species and parasite stages from digitalized thin smears based on simple visual cues (shape, size, texture and colour). While the accuracy of a single on-line expert is far from perfect, a single parasite classification obtained by combining the opinions of multiple on-line volunteers over the same smear, could improve accuracy and reliability of Plasmodium species identification in remote malaria diagnosis.M.L. holds a postdoctoral Fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-16409). JMB thanks the support by MINECO through research Grants BIO2013-44565R and BIO2016-77430R. MP, SGC, DC and MLO work was supported by the Universidad Politécnica de Madrid (COOP-XVII-02), Madrid Regional Government (TOPUS S2013/MIT-3024), the European Regional Development Funds, Amazon Web Services and Fundación Renta CorporaciónS

Surveillance of imported malaria in Spain: The useful tool of the Semi-Nested Multiplex PCR

The use of a new PCR-based method for the diagnosis of malaria in the Spanish Malaria Reference Laboratory has promoted an increase in confirmed cases of malaria. From August 1997 to July 1998, a total of 192 whole-blood samples and 71 serum samples from 168 patients were received from the hospitals of the Spanish National Health System. Most of the patients came from west-central African countries (85%). This molecular method showed more sensitivity and specificity than microscopy, detecting 12.4% more positive samples than microscopy and 13% of mixed infections undetectable by Giemsa stain. Plasmodium falciparum was the main species detected, with 68% of the total positive malaria cases, followed by Plasmodium malariae (29%), Plasmodium vivax (14%), and Plasmodium ovale (7%), including mixed infections in all cases. This report consists of the first wide, centralized survey of malaria surveillance in Spain. The reference laboratory conducted the analysis of all imported cases in order to detect trends in acquisition. The use of a seminested multiplex PCR permitted confirmation of the origins of the infections and the Plasmodium species involved and confirmation of the effectiveness of drug treatments. This PCR also allowed the detection of the presence in Spain of primaquine-tolerant P. vivax strains from west-central Africa, as well as the detection of a P. falciparum infection induced by transfusion.This work was supported by the Fondo de Investigaciones Sanitarias (FIS) (contract number 96/0216) and the Spanish Agency of International Cooperation (AECI). J. M. Rubio was granted a postdoctoral fellowship from the Comunidad Autónoma de Madrid, Madrid, Spain. J. Alvar was supported by a B.A.E. from the FIS (contract number 99/5038) and by the Christ’s College, University of Cambridge, Cambridge, United Kingdo

Molecular Diagnosis of Leishmaniasis in Spain: Development and Validation of Ready-To-Use Gel-Form Nested and Real-Time PCRs To Detect Leishmania spp

Leishmaniasis is an endemic parasitic disease in at least 98 countries. In Spain, it is considered a zoonosis caused by Leishmania infantum, with an annual incidence of 0.62 cases/100,000 inhabitants. The predominant clinical manifestations are the cutaneous (CL) and visceral forms (VL), and the diagnosis is performed by parasitological, serological, and molecular tests. At the WHO Collaborating Center for Leishmaniasis (WHOCCLeish), routine diagnostic tests are based on a nested PCR (Ln-PCR), culture, and serological tests. To simplify our PCR protocol, we aimed to develop and validate a ready-to-use nested gel-form PCR (LeishGelPCR) and a duplex real-time PCR (qPCR) that allowed simultaneous detection of Leishmania and mammalian DNA as an internal control (Leish-qPCR). Clinical validation was performed in 200 samples from the WHOCCLeish collection; 92 and 85 out of 94 and 87 samples were positive by LeishGelPCR and Leish-qPCR, respectively, showing a sensitivity of 98% in both approaches. The specificity was 100% for LeishGelPCR and 98% for Leish-qPCR. The limits of detection of both protocols were similar (0.5 and 0.2 parasites/reaction). Parasite loads in VL and CL forms were similar, although high loads were observed when invasive samples were tested. In conclusion, LeishGelPCR and Leish-qPCR showed excellent performance in the diagnosis of leishmaniasis. These new forms of 18S rRNA gene PCR are equivalent to Ln-PCR and can be introduced in the algorithm for CL and VL diagnosis. IMPORTANCE Although the gold standard for diagnosis of leishmaniasis is the microscopic observation of amastigotes, molecular techniques are becoming a cost-efficient alternative. Currently, PCR is a routine resource that is used in many reference microbiology laboratories. In this article, we have described two ways to improve the reproducibility and usability of the molecular detection of Leishmania spp. These new approaches could be introduced even in middle- and low-resource laboratories; one is a ready-to-use gel-form system of a nested PCR and the other is a real-time PCR. We show why molecular diagnosis is the best methodology to confirm a clinical suspicion of leishmaniasis with higher sensitivity than traditional methods, thus facilitating early diagnosis and timely treatment of human leishmaniasis.This research was supported by Subprograma Retos de Colaboración, Ministerio de Economía y Competitividad (RTC-2016-5245-1) and Agreement CNM-Mundo Sano Foundation-Spain (MVP 1379).S